Geographic Variation in Morphological, Genetic and Clonal Diversity of The
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Geographic variation in morphological, genetic and clonal diversity of the parasitoid wasp Pelecinus polyturator (Hymenoptera: Pelecinidae) By Victor Shegelski ENSC 489 Prepared for Dr. D. Hemmerling 04/04/2014 Abstract The wasp species Pelecinus polyturator, the pelecinid wasp, is a widely dispersed species that is most easily recognized by its abnormally long abdomen. The range of this organism’s habitat extends from Argentina to Canada making it subject to a wide range of weather and climatic conditions. This range in environmental variation could lead to adaptations including altered morphology and unorthodox reproductive techniques such as parthenogenesis. Samples have been collected from populations in Canada, the USA and Honduras and have been compared for geographic differences in morphology and genetic relatedness. Morphological comparisons show that: 1) Specimens from Canada and the USA are larger than Honduran specimens; 2) Honduran Specimens have relatively longer antennae; 3) American specimens have relatively longer abdomens; 4) Canadian specimens have less variance in wing size and antennae length. ISSR-PCR and agarose gel electrophoresis produced DNA bands based on the CA10 and GT10 primers that were used; however, the resolution was too low to be analysed for genetic relatedness. With methodological adjustments these primers are likely to produce significant results that can identify relatedness within and between populations. Keywords: Pelecinus polyturator, morphological variation, Darwin’s fecundity advantage model, Bergmann’s rule, selective pressure, climate change, climatic variation, population relatedness, thelytoky parthenogenesis, ISSR-PCR, Pelecinidae, Hymenoptera. Introduction Pelecinus polyturator (Drury) is an endoparasitoid wasp species that is primarily known for its incredibly long and thin abdomen with ovipositor as well as the parasitic larval stage in its life cycle (Brues, 1928). Sexual reproduction is rare due to low male representation in the species (Johnson and Musetti, 1999). P. polyturator compensates for the lack of sexual partners by performing thelytoky Shegelski 1 parthenogenesis (Brues, 1928); the female lays unfertilized eggs that produce clones of the mother. Extant species of these parasitoid wasps range from South Eastern Canada (as far West as Manitoba), through the eastern U.S.A. and the Honduran rain forests to Argentina (Johnson and Musetti, 1999). This study is designed to find relationships in the form of geographic trends in morphology, genetic diversity and clone presence in populations. Adult P.polyturator primarily use Phyllophaga spp. grubs as the host for their larvae (Johnson and Musetti, 1999); the wasp pierces the ground with its abdomen (up to 5 cm) in order to lay its eggs on beetle grubs, generally one per grub (Aguiar, 1997). Once the eggs on the grubs hatch, the parasitic wasp larvae enter the Phyllophagas host. The larvae feed and develop within the host and emerge to pupate; they eventually go through complete metamorphosis to emerge as an adult and, once an adult, the wasp feeds on nectar (Shih et al, 2009). The rarity of males is proportional to latitudinal positioning; Northern populations will have very few males (Aguiar, 1997) whereas the male presence in equatorial populations tends to be greater (Masner, 1995). Parthenogenesis is a form of reproduction in areas populated with males but the sexual reproduction also diversifies the clonal base of the community, as Vorburger (2006) found when studying parthenogenic aphids (Myzus persicae). One would expect lower genetic diversity in the Northern populations of P. polyturator as thelytoky parthenogenesis is the more common method of reproduction; however, Loxdale and Lushai (2003) found in some species (most prominently aphids) even cloned populations may still have a disparate gene pool by means of high mutation rates. With thelytokus and clonal diversity a species could easily colonize new locations and start new populations from a single individual (Loxdale and Lushai, 2003); P. polyturator have the largest distribution of the Pelecinidae (Johnson and Musetti, 1999) which may ultimately be due to their thelytoky parthenogenesis. Shegelski 2 Because of its vast geographic distribution, P. polyturator is subject to varying environmental conditions such as climate, food, host sources, predators and availability of sexual reproduction. It is likely that these varying conditions and pockets of sexual reproduction could lead to varying genetic diversity when measured geographically; this is especially possible considering there are gaps in the distribution of P.polyturator, effectively creating large ‘island’ populations (Johnson and Musetti, 1999). Female P. polyturator body length on average can range from 20-90mm and males average 15mm (Johnson and Musetti, 1999). A factor that could affect a size trend in P. polyturator may be host larvae size differences in which larger larvae lead to larger wasps (Johnson and Musetti, 1999); as mentioned above, host species of P. polyturator are primarily of the diverse genus Phyllophaga which are widely distributed and can also be found in the southern areas of Canada where P. polyturator exist (Galloway, 2008). The larvae of Phylophaga spp. can range from 15-25mm in length and current known larval hosts exploited by P. polyturator include Phyllophaga anxia (LeConte), P. inverse (Horn), P. drakei (Kirby), P. rugosa (Melsheimer) and Podischnus agenor (Olivier) (Johnson and Musetti 1999). In their research, Johnson and Musetti (1999) completed a large scale sample collection and comparison based on species variation, color and location collected. They also projected the regions of the Americas with an appropriate climate for the survival of P. polyturator . This area is much larger than its current dispersal and it has already been found thriving in areas even further north than what was projected (Galloway 2008) with no reported adverse effects on the ecosystem. This species is capable of adaptation that was never before predicted and the variety of grubs capable of acting as hosts for the pelecinid wasp is still not known. Clausen (1940) believed the original host of P. polyturator was actually a wood boring beetle similar to the mountain pine beetle (Dendroctonus ponderosae) currently ravaging the forests of Northern Alberta; given the climate projections by Johnson and Musetti (1999) it is possible that in the future this wasp could play a very beneficial role in Shegelski 3 agriculture and forestry as other parasitoid wasp species do today through biological pest control (Hoffmann and Frodsham 1993). Further analysis of size differences based on environmental factors and comparisons of genetic diversity have yet to be examined for P. polyturator. There are obvious differences in wing size between other parasitoid wasp species based upon flight requirements such as distance and power needed to get to food sources and hosts (Price, 1973); for example, if host populations are dense the wing size may be smaller as the energy is no longer required for long, frequent flights and would be best invested elsewhere. In Drosophila melanogaster, Frazer et al (2008) found that larger wing sizes can offer a biomechanical advantage in cold weather flying. It is possible the long thin abdomen with ovipositor could be more structurally robust in areas with heavy soil that requires more strength to get through to host grubs. In other Hymenoptera, specifically the Apidae species Bombus terrestris, or bumblebees, antennae length has been found to be related to olfactory sensitivity and detection threshold; longer antennae allow for more complex detection, and also lower the olfactory detection threshold (Spaethe et al 2007). Any consistent morphological variation between regions could reveal adaptive changes in the species, and it is possible that these regional adaptations could be the early stages of speciation through the founder effect (Templeton 1980). In order to examine genetic diversity intersimple sequence repeat-polymerase chain reaction (ISSR-PCR) was used as a form of genetic fingerprinting that targets base pair repetitions in the DNA of the P. polyturator specimens; this would help determine the clonal nature of geographic populations. In this case ISSR-PCR was used because there were no previously determined genetic markers for any Pelecinus spp. using the Scopus, Jstor and Springer Link databases, and using microsatallite markers is efficient but species specific (Selkoe and Toonen 2006). Without prior research, microsatellite determination is beyond the scope of this study. Shegelski 4 Selection of ISSR-PCR primers was ultimately be based on testing with a slightly defined starting point. Luque et al (2002) wanted to find genetic variation between different noctuids (from the Order Lepidoptera); they found in the ISSR-PCR testing of 11 primers that (CA)10 was the most accurate given a consistent 300ng of DNA was used in each test. Nascimento et al (2010) and Santos et al (2011) both tested 93 primers from UBC primer set #9 when testing genetic diversity and structure in bees from the Hymenoptera order. In each study (CA)n primers were among those selected for the final analysis. Uesugi and Sato (2012) found 3 (CA)n primers, (CA)11, (CA)12 and (CA)15, where among the 9 most efficient in their study isolating microsatellite loci in Encarsia smithi, a parasitoid wasp from the family Aphelinidae (order: Hymenoptera). Lucas et al (2009) identified several (GT)n